Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 https://doi.org/10.1186/s13023-021-02079-7 RESEARCH Open Access Considerations on diagnosis and surveillance measures of PTEN hamartoma tumor syndrome: clinical and genetic study in a series of Spanish patients Laura Pena‑Couso1, María Ercibengoa2, Fátima Mercadillo1, David Gómez‑Sánchez3,4, Lucía Inglada‑Pérez5, María Santos6, Javier Lanillos6, David Gutiérrez‑Abad7, Almudena Hernández8, Pablo Carbonell9, Rocío Letón6, Mercedes Robledo6,10, Cristina Rodríguez‑Antona6,10, José Perea11,12, Miguel Urioste1* and PHTS Working Group Abstract Background: The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish popula‑ tion and to identify new genes potentially associated with the disease. Results: We reviewed the clinical data collected through a specific questionnaire in a series of 145 Spanish patients with a phenotypic features compatible with PHTS and performed molecular characterization through several approaches including next generation sequencing and whole exome sequencing (WES). Macrocephaly, mucocuta‑ neous lesions, gastrointestinal polyposis and obesity are prevalent phenotypic features in PHTS and help predict the presence of a PTEN germline variant in our population. We also find that PHTS patients are at risk to develop cancer in childhood or adolescence. Furthermore, we observe a high frequency of variants in exon 1 of PTEN, which are associ‑ ated with renal cancer and overexpression of KLLN and PTEN. Moreover, WES revealed variants in genes like NEDD4 that merit further research. Conclusions: This study expands previously reported findings in other PHTS population studies and makes new contributions regarding clinical and molecular aspects of PHTS, which are useful for translation to the clinic and for new research lines. Keywords: PTEN hamartoma tumor syndrome, Cowden syndrome, PTEN gene, NGS, Exome Background The PTEN hamartoma tumor syndrome (PHTS; MIM 158350) encompasses several clinical entities with over- lapping phenotypic characteristics, that are associated with germline pathogenic variants in the PTEN gene. *Correspondence: murioste@cnio.es 1 Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba Familial Cancer Clinical Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain syndrome (BRRS) are the 2 principal entities of PHTS [1]. Full list of author information is available at the end of the article In addition to these, PHTS also includes some cases of A list of the group authors and their affiliations is provided in the Proteus-like syndromes, autism spectrum disorder (ASD) Additional file 2: Appendix S1. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 2 of 11 associated with macrocephaly, and VATER syndrome novo (24% of the PTEN variant carriers) and 7 familial [2–5]. Macrocephaly, mucocutaneous lesions, Lhermitte- variant cases (12%). Duclos disease (LDD) and hamartomatous polyposis are We will refer to carriers of PTEN pathogenic variants generally considered characteristic features of PHTS. and large deletions as PTEN-mut, excluding VUS car- Heterozygous germline variants in the tumor suppressor riers. The pathogenic variants appeared at higher fre- gene PTEN cause PHTS. These variants are inherited in quencies in exons 5 to 8 of PTEN, but we also found a an autosomal dominant pattern and are found in up to considerable number of patients carrying pathogenic 80–85% of CS patients, and in 60–65% of BRRS patients variants in exon 1 (Fig. 1A). Several types of vari- [2]. PTEN encodes a lipid and protein phosphatase that ants were observed (Fig. 1A) that affected the different among other functions antagonizes the proliferative PTEN protein domains, except the C-terminal end (resi- PI3K/AKT/mTOR pathway [6]. dues 352 to 403) (Fig. 1B). Some pathogenic variants in Besides numerous non-neoplastic multisystemic fea- PTEN appeared recurrently in our series: c.1003C > T, tures, PHTS also entails increased risks for thyroid, p.(Arg335*) (n = 5 unrelated patients); c.388C > T, breast, endometrial, colon, renal cell, and melanoma can- p.(Arg130*) (n = 3); c.39_40del, p.(Arg14Glufs*29) cers [7]. These high cancer risks reinforce the importance (n = 2); c.406T > C, p.(Cys136Arg) (n = 2); and c.697C > T, of an early diagnosis that allows appropriate surveillance p.(Arg233*) (n = 2). strategies. Reality, however, is quite the opposite: PHTS Array comparative genomic hybridization (aCGH) diagnosis usually takes a long time to be made, given the allowed exhaustive characterization of 5 large deletions rareness of the disease, its considerable clinical heteroge- which also encompassed the proximal gene KLLN. Two neity, and the lack of awareness and the limited knowl- of these rearrangements involved regions of 8 and 10 Mb, edge of this entity based on scarce studies. To improve affecting other genes such as BMPR1A (Fig. 1C, Addi- this situation, several diagnostic criteria and recommen- tional file 1: Table S3). dations have been proposed in the last decade [7–10]. The remaining 79 probands (54%) were negative for However, these recommendations are based on a lim- pathogenic variants, large rearrangements or VUS in the ited number of patients from only a few populations and PTEN gene. We will refer to these patients as PTEN-wt. there is still no consensus regarding their clinical and diagnostic utility [7, 9]. Clinical characterization and criteria for PTEN study Furthermore, PTEN germline variants are not detected The series of probands consisted mostly in adults (84%) in a considerable number of patients who meet the clini- but also included 25 patients (16%) under the age of cal diagnostic criteria [2], reason why multiple efforts 18 years old. 59% of the adults were women and 25% have been made to identify other genes involved in these men, plus 12% young men and 4% young women. In the syndromes. Variants in SDH-B, SDH-D, PIK3CA, AKT1, series of PTEN-mut+—excluding VUS—there were 32 TTN and SEC23B, together with hypermethylation of the women and 27 men. Mean age was 30 years in the muta- KLLN promoter, have been previously reported in certain tion carriers (PTEN-mut+) and 46 in the PTEN-wt. CS, CS-like and BRRS patients [11–16]. Excluding the PTEN VUS carriers (described in Addi- Here, we describe the clinical and molecular aspects of tional file 1: Table S2), we compared the phenotype 145 Spanish patients with a phenotype compatible with between the PTEN-mut and the PTEN-wt groups, and PHTS entities, to contribute to the knowledge of this rare found several clinical features that were significantly disease by defining its characteristics in a new population more common among PTEN-mut individuals, such as and searching for other relevant genes. macrocephaly, mucocutaneous lesions and obesity (body mass index ≥ 30), suggesting their usefulness as criteria to Results identify PTEN pathogenic variant carriers (Fig. 2A). On Spectrum of alterations in PTEN the other hand, other classical clinical features of PHTS, Almost half of the patients (46% of the total series) had a such as LDD, ASD and vascular lesions, did not signifi- germline alteration in the PTEN gene: 52 patients (36%) cantly discriminate between PTEN-mut and PTEN-wt carried pathogenic point variants, 7 individuals (5%) had individuals (Fig. 2A), suggesting they are poorer indica- large deletions and 7 additional patients (5%) carried var- tors of an individual carrying a PTEN pathogenic variant. iants of unknown significance (VUS). The complete list This was supported by regression analyses (Additional of PTEN alterations is shown in Additional file 1: Tables file 1: Table S4). S1-S3. No pathogenic variants nor VUS were found in the The incidence of cancer was higher in the PTEN-wt promoter of PTEN. We were able to trace the origin of compared to the PTEN-mut patients (58% (n = 46) ver- the PTEN variants in 21 cases through genetic testing of sus 39% (n = 23); p = 0.0017). The 3 most frequent can- their relatives. Thus, we confirmed the presence of 14 de cer types in the series (excluding the VUS carriers) were Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 3 of 11 A B 14 Catalytic 12 Number of pathogenic core 10 variant carriers 11% 8 6 Phosphatase 47% 4 C2 42% 2 0 Exon Exon Exon Exon Exon Exon Exon Exon Exon 1 2 3 4 5 6 7 8 9 Frameshift Missense Nonsense Splicing 37% 23% 29% 11% Intronic variants C BMPR1A KLLN PTEN Entire 1 1 2 3 4 5 6 7 8 9 gene Fig. 1 PTEN pathogenic variants found in our series. A PTEN point variants: numbers, distribution and types. B PTEN protein domains affected by the exonic variants. C Schematic representation of the gene regions affected by each of the 7 large deletions involving PTEN thyroid, colorectal and renal cancer, while breast, endo- of the PTEN-wt individuals; p = 0.013) developed cancer metrial and ovarian cancers were the most frequent in childhood or adolescence (Table 1). Some of the can- sex-linked cancers (Fig. 2B,C). Of the 69 cancer cases, cer types we encountered are very rare in this age range, 64 individuals had suffered some cancer within the spec- such as endometrial carcinoma or clear cell renal cell car- trum of PHTS (PHTS-associated cancer): 20 PTEN-mut cinoma. This suggests a risk to develop cancer at a very (31% of the PHTS-associated cancer patients) and 44 early age for carriers of germline pathogenic variants in PTEN-wt (69%). Nevertheless, we also found other can- PTEN. cer types not so frequently associated with PHTS, like Hodgkin lymphoma, meningioma and astrocytoma in Genotype–phenotype correlations PTEN-mut patients (Fig. 2B, C). In order to search for genotype–phenotype correlations, We noticed that 18 patients of our series were referred we evaluated the clinical features as a function of the for presenting only PHTS-associated cancers, with location of the variants along the PTEN sequence. We apparently no other feature of the disease. None of these found several associations, most notably an association individuals were carriers of PTEN pathogenic variants between renal cancer and pathogenic variants in PTEN and they accounted for 23% of the PTEN-wt patients, exon 1 (p = 0.045; Additional file 1: Table S6). suggesting that only the presentation of certain types of cancer might not be sufficient criterion to perform PTEN Search for other genetic factors genetic testing. This is also supported by regression anal- Given that not all patients with a clinical phenotype com- yses, which shows a poor contribution of the presence of patible with the PHTS entities are found to carry PTEN cancer alone to the probability of finding a PTEN patho- alterations, we searched for other genetic factors that genic variant in the patient (Additional file 1: Table S5). could be involved as causal elements in these PTEN- Interestingly, we found that a considerable propor- wildtype patients. Considering the large variability of tion of our patients (14% of the PTEN-mut and only 3% phenotypes found along the PHTS spectrum, we also Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 4 of 11 A Mucocutaneous (any) **** Macrocephaly **** Obesity **** Macular pigmentation penis **** Oral papillomas *** Gastrointestinal polyps *** Palmoplantar keratoses *** Musculoskeletal lesions ** Thyroid lesions ** Trichilemmomas ** Gynecologic lesions * Mammary lesions Lipomas or lipomatosis Glycogenic acanthosis Cutaneous neuromas Vascular lesions Mental retardation Lhermitte-Duclos disease Autism spectrum disorder Brain hamartomas Papules 0 10 20 30 40 50 60 70 80 90 100 Proportion of patients (%) Total series PTEN-wt PTEN-mut B Thyroid Renal Colorectal C Melanoma Breast *** Meningioma Endometrial Hodgkin lymphoma Astrocytoma Ovarian Bladder Testicular Ureter Prostate Lung adenocarcinoma 0 5 10 15 20 25 30 35 40 Thymus Endocrine Proportion of patients (%) 0 5 10 15 20 25 Total series PTEN-wt PTEN-mut Proportion of patients (%) Total series PTEN-wt PTEN-mut Fig. 2 Proportion of individuals showing the indicated clinical manifestations in our series. A Occurrence of benign clinical features. B Occurrence of the different cancer types (not linked to sex). C Occurrence of sex-linked cancer types. PHTS-associated cancer types are boxed. Chi-square or Fisher test significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) is shown for comparisons of absolute numbers of the PTEN-wt and PTEN-mut groups searched for genes that could be involved as phenotype of KLLN was similar in patients and controls, except for modifiers. patients carrying PTEN variants in exon 1, who showed We first studied the mRNA expression of KLLN in our overexpression of KLLN (Fig. 3A). These individuals patient series. Strikingly, we found that the expression also showed an unexpected high expression of PTEN Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 5 of 11 Table 1 Cancer occurrence in individuals ≤ 18 years old from our series. Each case corresponds to a unique proband Cancer type Number of probands Age of onset PTEN germline status Papillary thyroid cancer 1 16 p.(Lys6Argfs*4) 1 16 p.(Arg335*) Follicular thyroid cancer 1 14 p.(Arg335*) Endometrial adenocarcinoma 1 15 p.(Cys136Arg) Ovarian endodermal sinus tumor 1 6 p.(Cys136Arg) Clear cell renal cell carcinoma 1 18 p.0? Hodgkin lymphoma 1 18 p.(Gln17*) Testicular mixed germ cell tumor 1 18 p.(Thr277Asnfs*21) Hodgkin lymphoma 1 6 WT Thyroid cancer (unspecified type) 1 14 WT Fig. 3 mRNA expression analyses. Relative mRNA expression of KLLN (A), PTEN (B) and PTENP1 (C), determined by qPCR using 36B4 as a reference gene. Control individuals were compared to subgroups of PHTS patients according to the variant status of PTEN. Only significant differences are indicated (two tailed t-test; *p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001). Each dot represents the mean value of the expression of the gene under study for each patient (assessed in triplicate). The mean value of each group ± SEM is indicated (Fig. 3B), and we could rule out an upregulation of the the variants found (Additional file 1: Table S8) has been pseudogene PTENP1 (Fig. 3C). Therefore, only variants described to trigger the mTOR pathway and they were in exon 1 of PTEN seem to alter the expression levels of classified as VUS until additional experiments are per- PTEN and its neighbor gene KLLN, although it is unclear formed. Moreover, the number of variants in mTOR how this contributes to PHTS etiology or pathogenesis. pathway genes was similar in the groups of PTEN-mut The only distinctive clinical feature of the patients with and PTEN-wt patients, and we did not find any clear cor- KLLN and PTEN overexpression was an increased pres- relation between these variants and a specific phenotype ence of renal cancer, as noted above. in the carriers. The gene panel also targeted genes asso- We also searched for alterations in mTOR pathway ciated with other cancer predisposition syndromes (such genes that could be involved either as phenotype modi- as FLCN) and we found some PTEN-wt patients with fiers in the PTEN-mut patients or as causal factors in rare variants in these genes (Additional file 1: Table S8). the PTEN-wt patients. For this purpose, we screened This prompted us to consider a clinical reevaluation of almost our entire series (127 patients, including PTEN- these patients. mut and PTEN-wt) by using a multigene panel (see the In order to expand the search for genes involved in full list of genes in Additional file 1: Table S7). None of PHTS phenotypes other than PTEN and mTOR pathway Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 6 of 11 genes, we performed WES in 11 unrelated PTEN-wt description of their clinical picture. Understanding that patients selected for their clinical features (see Meth- the percentages of each clinical trait should be inter- ods) and we found several potentially relevant germline preted as a minimum estimate, we can draw some valu- variants (Additional file 1: Fig. S1). On the one hand, we able conclusions by comparing those patients with and found variants in known cancer genes (such as MUTYH, without mutation in the PTEN gene. associated with colorectal cancer risk) that might explain As expected, several clinical features such as macro- a certain feature of the patient’s phenotype, but not the cephaly and mucocutaneous lesions were significantly development of a syndrome that resembles PHTS. On more frequent among the PTEN pathogenic variant car- the other hand, we found several candidate variants to riers, suggesting their usefulness as clinical diagnostic be further assessed in following studies (e.g. in RNF135, criteria. Together with these features, another good indi- NEDD4 and HERC1). cator of the presence of a PTEN pathogenic variant was Additionally, available tumor DNAs were studied using obesity, present in 22% of our PTEN-mut individuals. the mentioned gene panel and the somatic variants found Obesity rates in the Spanish population were: men 15.1%, were in agreement with each cancer type, including a women 13.1%, boys 10.6%, girls 11.8% (Global Obesity splicing variant in PTEN in the lung adenocarcinoma Observatory, https://​data.​world​obesi​ty.​org/​count​r y/​ sample, and BRAF p.(V600E) and PIK3CA p.(G1049R) in spain-​199/). This feature was hardly noted in the other thyroid cancer samples (Additional file 1: Table S9). patient series described in the literature [7, 8, 10]. Obesity has a very heterogeneous origin, and high rates observed Discussion in our series could be related with some features of the Our study describes clinical and molecular findings in a syndrome (e.g. thyroid disorders), but the association of series of 145 patients with clinical features of PHTS enti- PTEN and obesity could also be related with the involve- ties, the largest one studied in the Spanish population ment of PTEN in the insulin pathway [21, 22]. Despite of thus far [17]. In agreement with previous studies in other the limitations of our sample size it is important to high- populations [7, 8, 10], the pathogenic variants found in light this finding since obesity is a known risk factor for our series were located along the PTEN sequence, with cancer, with a strong causal link for breast, uterine, colon hotspots in exons 5 and 8. In contrast to those other and renal cancers [23], and we observed that half (6 out studies, however, we found a relatively high number of of 13) of the obese PTEN-mut patients suffered cancer individuals with pathogenic variants in exon 1 [7, 8, 10]. with a median onset age of 22 years old (versus 33 for All variants in exon 1 differed from each other, ruling out non-obese patients). We therefore suggest that attention a founder effect. Our sample size does not allow us to should be given to obesity in the surveillance of PHTS support the conclusion that this is a specific characteris- patients. In fact, it might be advisable to consider PI3K tic of Spanish PHTS patients. The study of more individ- inhibitors in the treatment of PHTS patients not only to uals in other populations and in ours is required to better reverse classical PHTS lesions (such as skin hamartomas) address this hypothesis. [24], but also to treat obesity [25, 26]. When analyzing large rearrangements, we found On the other hand, some characteristics classically several cases in which the deletion also affected other associated with CS, such as LDD (OR = 3.78, p = 0.11) genes, such as KLLN, a neighbor gene of PTEN, but also and the presence of cancer (OR = 0.40, p = 0.05) alone, BMPR1A, a further upstream gene associated with juve- were poor predictors of a PTEN pathogenic variant. In nile polyposis syndrome (JPS; MIM 174,900) and colo- the present study, we noticed that many clinicians find rectal cancer risk. Hence, the 2 patients with a deletion the presence of 2 or more CS-associated cancers a suf- affecting both PTEN and BMPR1A showed clinical fea- ficient diagnostic criterion to refer a patient for PTEN tures of both PHTS and JPS. This suggests an additive testing. However, we found that all patients with CS- effect of the two genes and a possibly increased risk of associated cancers but without any other reported colorectal cancer in these individuals. This finding is not clinical feature of CS (such as macrocephaly or mucocu- uncommon in PHTS patients, as it has been previously taneous lesions) were PTEN-wt, i.e., none of them car- reported [18–20]. Therefore, we recommend to extend ried an alteration in PTEN. Therefore, the presence of the study of large deletion carriers by using methods such CS-associated cancers should not be criterion to refer a as aCGH or gene panels that allow copy number variant patient for PTEN testing, unless this is accompanied by identification, in order to identify other genes that might other features of the disease. also be deleted and cause additional clinical risks for the Cancer has been usually described to develop dur- patient. ing adulthood in PTEN pathogenic variant carriers [7, Our series of patients comes from very different medi- 8, 10], and only one study highlighted the risk of early cal specialists and not in all cases we have an exhaustive cancer development, especially thyroid cancer [27]. In Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 7 of 11 fact, the guidelines of the U.S. National Comprehensive a probably pathogenic variant in FLCN, a gene that is Cancer Network already suggest a yearly thyroid ultra- associated with the Birt-Hogg-Dubé (BHD) syndrome sound for PHTS patients under 18 [28]. Based on our which includes cutaneous lesions that can resemble CS findings, 8 patients developed cancer under 18 among 59 lesions. This example suggests the need for perform- patients PTEN-mut+, (Table 1), if a cancer is diagnosed ing a differential diagnosis considering syndromes that at young age, it should be consider also genetic testing for have overlapping clinical features with the PHTS enti- PTEN variants. A large prospective study in young PHTS ties. We also performed WES and found some interest- patients would be useful to establish the appropriate age ing variants that could account for etiological factors in at which screening should begin for each cancer type in PTEN-wt patients: variants in RNF135, associated with these individuals. overgrowth, macrocephaly and facial dysmorphism We explored if the different clinical diagnostic crite- [29]; variants in UBN2, associated with autism [30]; ria proposed in the literature [7, 9] could retrospectively and variants in NEDD4 and HERC1, which encode two identify the patients from our series (Additional file 1: ubiquitin ligases involved in PTEN and TSC2 degrada- Fig. S2). The International Cowden Consortium (ICC) tion, respectively [31, 32]. We did not find candidate and the Cleveland Clinic (CC) score had a good per- variants in SDH-B, SDH-D, PIK3CA, AKT1, TTN or formance by identifying around 90% of our PTEN-mut SEC23B; these genes were suggested in the literature to patients (as expected, given the similarity of our criteria be involved in CS and BRRS [11–14]. to these), together with 40% of the PTEN-wt patients. Several LDD patients do not present PTEN alterations By contrast, when using the revised criteria proposed and recently EGFR was proposed as a novel candidate by Pilarski et al. [9] we failed to identify a considerable for LDD susceptibility [33, 34]. Thus, we also sought for amount of the patients carrying PTEN pathogenic vari- other LDD genes through WES in 4 PTEN-wt patients ants (more than 50% of these individuals). with this cerebellar tumor. We did not find any relevant To date, there are no strong genotype–phenotype cor- alterations in EGFR, but of note, we found an as yet relations in PHTS, mainly due to the small sample sizes unreported variant in FGFR1, which encodes a receptor of the studies. Bearing in mind the limitations due to involved in PI3K signaling [35, 36]. the size of our cohort, we did find some significant asso- ASD has a complex etiology, with at least 1,000 suscep- ciations between the location of the PTEN pathogenic tibility genes reported [30] and variants in PTEN account variant and the phenotype. Interestingly, we observed for a relevant amount of individuals with ASD and mac- an association between renal cancer and PTEN exon 1 rocephaly [37]. Five PTEN-wt patients of our series variants (p = 0.045). As KLLN was described as a possi- showed this phenotype. Through WES in one of these ble phenotype modifier [15], we explored its role in our individuals, we found a missense variant in the ATR​gene PHTS patients and found an overexpression of KLLN (malfunction of its protein can impair fragile site stabil- that correlated with an overexpression of PTEN (which ity, which can be a risk for autism), a stop gain variant may indicate a co-regulation of these genes due to their in UBN2 and two different variants in EP400; variants in shared bidirectional promoter). However, no allelic– the latter two genes have been suggested to be associated specific expression studies have been done, so we can´t with autism [30]. determine if the upregulation is limited to the mutated We found several variants in other genes besides PTEN allele. In consequence, the mechanism underlying this that could account for a subset of the patients who tested observation and its hypothetical association with an negative for alterations in PTEN, but their importance increase in risk for renal cancer in the individuals that remains to be determined before a translation to the clin- harbored variants in PTEN exon 1, is currently unclear. ical setting can be considered. Moreover, we did not find Since a considerable proportion of CS, CS-like, a common gene altered in several patients, similar to the BRRS and ASD-macrocephaly patients (from 20 to results of other authors [38, 39], and only certain gene 90%, depending on the clinical entity) do not carry a variants could explain specific individual cases. There- PTEN germline pathogenic variant [2–5], it is relevant fore, it is possible that PTEN is the only high suscepti- to continue searching for new genetic factors involved bility gene of CS, CS-like, BRRS or ASD-macrocephaly, in PHTS development, to improve counseling, risk and other yet to be discovered factors might explain the assessment and therapeutic measures for each patient. disease in individuals with no variants in PTEN. Other Even though we did not find any clear candidate, sev- approaches such as RNA-sequencing, genome sequenc- eral variants in known cancer predisposition genes ing or methylation assays might shed light on this issue. (such as FLCN, MUTYH and BAP1) were observed, The fact that the somatic variants found in our work which made us reconsider the clinical diagnosis of in the cancer tissues were to be expected for each can- these patients. As an example, one patient harbored cer type could point towards a similar evolution pattern Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 8 of 11 of tumors in PHTS and their sporadic counterparts, PTEN genetic analyses although we were only able to study a small sample set. DNA was extracted from samples of peripheral blood leukocytes of each proband. The presence of ger- Conclusions mline variants was evaluated in all 9 exons of PTEN Our findings suggest that to improve diagnosis, focus (together with the intron–exon boundaries) by PCR and should be put on macrocephaly, mucocutaneous lesions, Sanger sequencing. Variants were named in relation to obesity and gastrointestinal polyposis when performing NM_000314.4. Positive results were confirmed in a sec- the clinical evaluation, as these were features that best ond blood sample using the MyTaqBlood PCR (Bioline) suggested the presence of a PTEN pathogenic variant. reagent. Large rearrangement analysis was performed Once the PTEN germline status of the patient is known, through multiplex ligation-dependent probe amplifica- it is relevant to perform a differential diagnosis in case tion (MLPA) with SALSA P225D1 (MRC Holland). The no pathogenic variants were found. In this last scenario SurePrint G3 Unrestricted CGH 4x180K microarray or when finding large rearrangements, it is important to (Agilent) was used for cases with large deletions involv- expand the search to other genes that might be altered ing the 5’ end of PTEN to interrogate the extent of the causing additional or unexpected clinical risks. Finally, deletion and the location of the breakpoints. The PTEN regarding the management and follow-up recommenda- promoter was analyzed in 31 patients in whom no poten- tions for PHTS, we suggest regularly monitoring weight tially relevant changes in the PTEN gene were detected. and considering cancer screenings at an earlier age in Primers are mentioned in Additional file 5: Methods S2. young individuals. Prospective studies of PHTS patients will aid in the determination of their clinical risks. mRNA expression analyses RNA was extracted from peripheral blood leukocytes Materials and methods with TRIzol reagent (Thermo Fisher). Concentration Patients and clinical evaluation and integrity of the RNA were checked using NanoDrop One hundred and forty-five probands (unrelated individ- (ND-1000 V3.7.1; Thermo Fisher). The High Capacity uals from unique families) meeting relaxed clinical crite- cDNA reverse transcriptase kit (Applied Biosystems) ria from the International Cowden Consortium (ICC) [7], was used to synthesize cDNAs. cDNAs from unaffected including patients who a) met the pathognomonic cri- donors were used as controls for expression levels in the teria, b) met 1 major criterion and 2 minor criteria, and qPCR reaction. Samples (n = 23) and controls (n = 23) c) suffered any 2 of the following cancer types within the were analyzed in triplicate. Primers for PTEN, KLLN and GoTaq® qPCR Master Mix (Promega) was used for the PHTS spectrum: breast, thyroid or endometrial cancer, PTENP1 are listed in Additional file 4: Methods S1. The were included in our series. All patients were Caucasians of Spanish origin (age range from 1 to 76 years old; 84% reaction and the qPCR was performed in an ABI Quant- adults and 16% ≤ 18 years old). 26 patients were seen in Studio S6 Flex System (Applied Biosystems). 36B4 was the consultancy of our group at the University Hospital used as the reference gene to calculate relative mRNA of Fuenlabrada (UHF, Madrid, Spain). The remainder 119 ­ −∆∆Ct method for qPCR analysis. expression using the 2 patients were referred to our laboratory through collabo- ration with medical specialists of 35 different hospitals Next generation sequencing (NGS) panel from Spain (PHTS Working Group). Biological samples, A total of 131 DNA samples (127 from blood and 4 from clinical information and signed informed consent were paraffin-embedded tumor tissue samples) were included referred to our laboratory at the Familial Cancer Clinical in a custom NGS panel from NimbleGen (Roche) to look Unit (CNIO, Madrid, Spain). Written formal consent was for other possible genetic factors involved as phenotype obtained from the parents or guardians of individuals modifiers or with a causal role. Variant filtering consisted under 18 years old. The patients’ phenotype information of maintaining only the variants in canonical transcripts was collected through a clinical questionnaire specifi- (APPRIS) with high or moderate functional effect—refer cally designed for this project (Additional file 4: Methods to the Variant Effect Predictor (VEP, Ensembl) calculated S1). We sent 119 questionnaires to medical profession- impacts, with moderate impact corresponding to cod- als from 9 different specialties. 51 questionnaires were ing non-synonymous variants (e.g. missense, in-frame returned to us completed. The project has the approval indels) and high to loss-of-function variants (e.g. non- of the Ethics Committee of the UHF (approval number: sense, those disrupting canonical splice sites, frameshift 20/28). indels)—, a variant allele frequency (VAF) between 0.3 and 0.6 for heterozygotes and > 0.9 for homozygotes, and a minor allele frequency (MAF) < 0.1% (gnomAD). Somatic variants were called with Mutect2 (GATK4, ref ) Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 9 of 11 and variants were filtered out following the following cri- esophageal and renal abnormalities; VUS: Variant of unknown significance; WES: Whole exome sequencing. teria: “LOW” and “MODIFIER” categories according to VEP; variants present in the matched-paired blood sam- ple (germline variants); variants with a VAF < 15% “ were Supplementary Information The online version contains supplementary material available at https://​doi.​ excluded. Further details are described in the Additional org/​10.​1186/​s13023-​021-​02079-7. file 4: Methods S1 and Additional file 1: Table S7. Additional file 1: Fig. S1. Clinical manifestations and selected gene variants for the 11 PTEN-wt patients studied through WES. Group A: Whole exome sequencing (WES) patients who meet strict Pilarski* diagnostic criteria; group B: patients who Eleven individuals with no pathogenic variants or vari- developed LDD but do not meet strict Pilarski* diagnostic criteria; group C: pediatric case with macrocephaly together with neurological alterations ants of unknown significance (VUS) identified in PTEN and overgrowth. *Pilarski et al. 2013. Fig. S2. Proportion of individuals in (7 meeting Pilarski’s clinical diagnostic criteria [9], 3 each group of our series identified using the indicated diagnostic criteria with LDD, and 1 pediatric case with macrocephaly, from the literature. (See references in Additional file 3: Appendix S2). Table S1. PTEN pathogenic point variants found in our series. Variants are autism and overgrowth) were selected for whole exome named in relation to NM_000314.4. Table S2. PTEN variants of unknown sequencing (WES). Germline genomic DNA samples significance (VUS) found in our series. Variants are named in relation to from these individuals were quantified using Quant-iT NM_000314.4. Table S3. Deleted chromosomal regions found in our series through MLPA and aCGH. Table S4. Risk of carrying a PTEN germline PicoGreen dsDNA reagent (Thermo Fisher), their qual- pathogenic variant according to different clinical features, based on ity was checked using the NanoDrop spectrophotometer analysis of our patient series. Odds ratios obtained with logistic regression. (ND-1000 V3.7.1; Thermo Fisher) and degradation was Table S5. Risk of carrying a PTEN germline pathogenic variant when the patient presents only a certain clinical feature. Odds ratios obtained with assessed by agarose gel electrophoresis. WES (12 Gb, logistic regression. Table S6. Genotype–phenotype associations found 100X coverage) and primary bioinformatics analysis in our series. Chi-square or t-test significance is shown. Table S7. List of were performed at the Novogene Bioinformatics Insti- genes included in the multigene panel. Table S8. Germline VUS found in mTOR-related genes by NGS. Table S9. Germline PTEN status and tute (Beijing, China). Further details are described in the NGS findings in the indicated tumor samples (each one from a different Additional file 4: Methods S1. proband). Additional file 2: Appendix S1. List of authors and affiliations of the PHTS working group. Interpretation and validation of variants Additional file 3: Appendix S2. References related to supporting Variants were considered deleterious if they were information. described as such in public databases (ClinVar, HGMD Additional file 4: Methods S1. Clinical questionnaire. and LOVD), or when the specific study of cDNA Additional file 5: Methods S2. Primers and further information regarding sequence supported a deleterious consequence. The pres- the NGS and WES. ence of variants was confirmed in a second sample using a different method: MyTaq Blood-PCR Kit (Bioline). Acknowledgements We thank the families and their caregivers for providing consent to participate in this study. Statistical analyses Authors’ contributions Chi-square or Fisher’s exact tests were used to evalu- LP-C collected, evaluated and interpreted clinical and molecular data. ME: recruited patients and revised the manuscript. FM, PC and RL: performed and ate differences between our cohort and other previously interpreted genetic studies. DG-S: analyzed results of WES. LI-P: performed published cohorts [7, 8, 10] using R. Logistic regression statistical analysis. MS, JL and CRA: evaluated and interpreted results of NGS was used to evaluate associations and risks using SPSS. panel. DG-A, AH and PHTS Working Group: cared and recruited patients. MR and JP: conceptualized the study, interpreted clinical and genetic data, qPCR analyses (t-test, Mann–Whitney) were done using reviewed and revised the manuscript. MU: cared and recruited patients, GraphPad Prism (GraphPad Software, Inc.). Bilateral p designed and supervised the study and reviewed the manuscript. All authors values < 0.05 were considered statistically significant. read and approved the final manuscript. Funding This work was supported by the Spanish Ministry of Economy and Com‑ Abbreviations petitiveness (MINECO) and the European Regional Development Fund in aCGH: Array comparative genomic hybridization; ASD: Autism spectrum the framework of project PI14/00459, and by a grant from the Federación disorder; BHD: Birt-Hogg-Dubé; BRRS: Bannayan-Riley-Ruvalcaba syndrome; Española de Enfermedades Raras (FEDER). FPI fellowship BES‐2015‐071383 to CC: Cleveland Clinic; CS: Cowden syndrome; HGMD: Human gene mutation LP-C, funded by MINECO and the European Social Fund (ESF). The funders had database; ICC: International Cowden Consortium; LDD: Lhermitte-Duclos no role in study design, data collection and analysis, decision to publish, or disease; JPS: Juvenile polyposis syndrome; LOVD: Leiden open variation data‑ preparation of the manuscript. base; MAF: Minor allele frequency; MLPA: Multiplex ligation-dependent probe amplification; PHTS: PTEN hamartoma tumor syndrome; NGS: Next genera‑ Availability of data and materials tion sequencing; VAF: Variant allele frequency; VATER: Vertebral, anal, tracheal, The dataset of current study are not publicly available but are available from the corresponding author on reasonable request. Pena‑Couso et al. Orphanet Journal of Rare Diseases (2022) 17:85 Page 10 of 11 Declarations 6. Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012;13:283–96. Ethical approval and consent for participation and publication 7. Tan M-H, Mester J, Peterson C, Yang Y, Chen J-L, Rybicki LA, et al. A clinical This study was approved by the research ethics committee of the University scoring system for selection of patients for PTEN mutation testing is pro‑ Hospital of Fuenlabrada (approval number 20/28). Parents or guardians of posed on the basis of a prospective study of 3042 probands. Am J Hum individuals under 18 years old signed written informed consents forms for the Genet. 2011;88:42–56. investigation and publication of articles. 8. Nieuwenhuis MH, Kets CM, Murphy-Ryan M, Yntema HG, Evans DG, Colas C, et al. Cancer risk and genotype–phenotype correlations in PTEN Consent for publication hamartoma tumor syndrome. Fam Cancer. 2014;13:57–63. Not applicable. 9. Pilarski R, Burt R, Kohlman W, Pho L, Shannon KM, Swisher E. Cowden syndrome and the PTEN hamartoma tumor syndrome: systematic review Competing interests and revised diagnostic criteria. J Natl Cancer Inst. 2013;105:1607–16. 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